CN113166221A - 制备在液体药物组合物中高度稳定的具有治疗活性的阿地白介素的方法 - Google Patents
制备在液体药物组合物中高度稳定的具有治疗活性的阿地白介素的方法 Download PDFInfo
- Publication number
- CN113166221A CN113166221A CN201980039901.2A CN201980039901A CN113166221A CN 113166221 A CN113166221 A CN 113166221A CN 201980039901 A CN201980039901 A CN 201980039901A CN 113166221 A CN113166221 A CN 113166221A
- Authority
- CN
- China
- Prior art keywords
- aldesleukin
- cell
- sds
- stable
- year
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108700025316 aldesleukin Proteins 0.000 title claims abstract description 88
- 229960005310 aldesleukin Drugs 0.000 title claims abstract description 80
- 239000007788 liquid Substances 0.000 title claims abstract description 17
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 8
- 238000004519 manufacturing process Methods 0.000 title description 17
- 238000000034 method Methods 0.000 claims abstract description 52
- 239000000203 mixture Substances 0.000 claims abstract description 31
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 9
- 201000011510 cancer Diseases 0.000 claims abstract description 7
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical group [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 42
- 210000004027 cell Anatomy 0.000 claims description 37
- 241000588724 Escherichia coli Species 0.000 claims description 14
- 238000003556 assay Methods 0.000 claims description 11
- 230000001225 therapeutic effect Effects 0.000 claims description 11
- 238000004587 chromatography analysis Methods 0.000 claims description 10
- 238000000338 in vitro Methods 0.000 claims description 9
- 210000003289 regulatory T cell Anatomy 0.000 claims description 9
- 238000002296 dynamic light scattering Methods 0.000 claims description 8
- 230000004071 biological effect Effects 0.000 claims description 7
- 238000009826 distribution Methods 0.000 claims description 7
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 239000013604 expression vector Substances 0.000 claims description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 238000004113 cell culture Methods 0.000 claims description 4
- 210000003000 inclusion body Anatomy 0.000 claims description 4
- 239000000919 ceramic Substances 0.000 claims description 3
- 239000003599 detergent Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 3
- 230000003647 oxidation Effects 0.000 claims description 3
- 238000007254 oxidation reaction Methods 0.000 claims description 3
- 230000001590 oxidative effect Effects 0.000 claims description 3
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 3
- 239000008365 aqueous carrier Substances 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 238000010171 animal model Methods 0.000 claims 2
- 230000006052 T cell proliferation Effects 0.000 claims 1
- 230000003381 solubilizing effect Effects 0.000 claims 1
- 238000011282 treatment Methods 0.000 abstract description 6
- 208000023275 Autoimmune disease Diseases 0.000 abstract 1
- 208000037979 autoimmune inflammatory disease Diseases 0.000 abstract 1
- 108010002350 Interleukin-2 Proteins 0.000 description 56
- 102000000588 Interleukin-2 Human genes 0.000 description 56
- 125000003275 alpha amino acid group Chemical group 0.000 description 16
- 230000000694 effects Effects 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 16
- 239000000243 solution Substances 0.000 description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 238000009472 formulation Methods 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 8
- 238000003860 storage Methods 0.000 description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 6
- 210000001744 T-lymphocyte Anatomy 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 101001002657 Homo sapiens Interleukin-2 Proteins 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 239000001488 sodium phosphate Substances 0.000 description 5
- 229910000162 sodium phosphate Inorganic materials 0.000 description 5
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 4
- 102000004127 Cytokines Human genes 0.000 description 4
- 108090000695 Cytokines Proteins 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000000855 fermentation Methods 0.000 description 4
- 230000004151 fermentation Effects 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 235000011008 sodium phosphates Nutrition 0.000 description 4
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 4
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 229930195725 Mannitol Natural products 0.000 description 3
- 206010027480 Metastatic malignant melanoma Diseases 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000011109 contamination Methods 0.000 description 3
- -1 copper chloride Chemical class 0.000 description 3
- 238000011026 diafiltration Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000000594 mannitol Substances 0.000 description 3
- 235000010355 mannitol Nutrition 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 208000021039 metastatic melanoma Diseases 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 238000007911 parenteral administration Methods 0.000 description 3
- 229940087463 proleukin Drugs 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 2
- IZHVBANLECCAGF-UHFFFAOYSA-N 2-hydroxy-3-(octadecanoyloxy)propyl octadecanoate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)COC(=O)CCCCCCCCCCCCCCCCC IZHVBANLECCAGF-UHFFFAOYSA-N 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 241000831652 Salinivibrio sharmensis Species 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108091008874 T cell receptors Proteins 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 238000002659 cell therapy Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 102000055277 human IL2 Human genes 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 239000003701 inert diluent Substances 0.000 description 2
- 230000028709 inflammatory response Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000012669 liquid formulation Substances 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000001788 mono and diglycerides of fatty acids Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 108010014312 DNA-Cytosine Methylases Proteins 0.000 description 1
- 102000016923 DNA-Cytosine Methylases Human genes 0.000 description 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 1
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 1
- 108010038453 Interleukin-2 Receptors Proteins 0.000 description 1
- 102000010789 Interleukin-2 Receptors Human genes 0.000 description 1
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 108050004171 Lon proteases Proteins 0.000 description 1
- 206010027458 Metastases to lung Diseases 0.000 description 1
- 206010050513 Metastatic renal cell carcinoma Diseases 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 108010008681 Type II Secretion Systems Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000003945 anionic surfactant Substances 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 239000003855 balanced salt solution Substances 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 238000002619 cancer immunotherapy Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000009172 cell transfer therapy Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000008094 contradictory effect Effects 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 229940061607 dibasic sodium phosphate Drugs 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 1
- 238000011143 downstream manufacturing Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229940074045 glyceryl distearate Drugs 0.000 description 1
- 229940075507 glyceryl monostearate Drugs 0.000 description 1
- 229960004931 histamine dihydrochloride Drugs 0.000 description 1
- PPZMYIBUHIPZOS-UHFFFAOYSA-N histamine dihydrochloride Chemical compound Cl.Cl.NCCC1=CN=CN1 PPZMYIBUHIPZOS-UHFFFAOYSA-N 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 238000012454 limulus amebocyte lysate test Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000011469 lymphodepleting chemotherapy Methods 0.000 description 1
- 239000012931 lyophilized formulation Substances 0.000 description 1
- 230000001320 lysogenic effect Effects 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 101150093139 ompT gene Proteins 0.000 description 1
- 108010003052 omptin outer membrane protease Proteins 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000003182 parenteral nutrition solution Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000027317 positive regulation of immune response Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- VBJGJHBYWREJQD-UHFFFAOYSA-M sodium;dihydrogen phosphate;dihydrate Chemical compound O.O.[Na+].OP(O)([O-])=O VBJGJHBYWREJQD-UHFFFAOYSA-M 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000012289 standard assay Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 239000008227 sterile water for injection Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000009827 uniform distribution Methods 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
- A61K38/2013—IL-2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/55—IL-2
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Toxicology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Diabetes (AREA)
- Inorganic Chemistry (AREA)
- Oncology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Pulmonology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Rheumatology (AREA)
Abstract
本发明涉及阿地白介素/SDS聚集体的液体药物组合物及其在治疗自身免疫性疾病、炎性病症和癌症中的用途。还描述了制备所述组合物的方法。
Description
相关申请的交叉引用
本申请要求2018年6月13日提交的美国临时申请No:62/684,288(其以引用的方式整体并入本文)在35 U.S.C.§119(e)下的优先权的权益。
技术领域
本发明涉及包含在溶液中稳定的阿地白介素/SDS聚集体的新药物组合物及其药物用途。
背景技术
白介素2(IL-2)是调节T细胞和NK细胞的存活、增殖和分化的关键细胞因子(1)。这些生物学活性的发现导致IL-2在1994年被批准用于转移性肾癌的癌症免疫治疗的临床应用,1998年被批准用于转移性黑素瘤的临床应用。此后,IL-2成为推动从患者的肿瘤中提取的T淋巴细胞(肿瘤浸润淋巴细胞,TIL)的体外扩增的试剂,所述T淋巴细胞随后转移回患者以避免未来癌症的发展(2)。最近,IL-2也已用于扩增用抗原特异性T细胞受体转导的T细胞(3)或用于癌症治疗的用嵌合抗原受体(CAR)转导的T细胞(4)。由于发现IL-2在免疫应答中具有以下双重功能,最近出现了IL-2医学应用的新领域:a)众所周知的作为炎性应答的活化剂的活性,以及b)作为炎性应答的下调因子诱导调节性T细胞(CD4+Foxp3+Treg细胞)扩增(5)。可通过两种具有高或低的亲和力的IL-2受体的存在以及它们的特定细胞分布来解释此种看似矛盾的双重性(5)。此种IL-2的双重性导致了如下所述的相反的疗法:a)如在最初的癌症治疗中一样以高剂量使用IL-2来刺激免疫应答,以及b)如在自身免疫或炎性病症的治疗中一样以低剂量使用IL-2来抑制过度或异常的免疫应答。
当前的IL-2的多数临床应用(如果不是全部的话)是使用称为阿地白介素(des-ala-2ser125)(PROLEUKINTM)的天然IL-2的重组突变形式进行的。美国专利4,604,377教导了如何制备在无菌、稳定的冻干配制物中始终稳定(consistent)的重组IL-2(包括阿地白介素)的药物组合物,其中重组IL-2与提供体积的水溶性载体(例如甘露醇)和足以确保重组IL-2在水中的溶解度的量的十二烷基硫酸钠混合。所述配制物适合在水性注射剂中复水,所述注射剂用于肠胃外施用,在人患者中稳定和耐受良好。另一方面,最近在欧洲专利EP 1,688,146B中公开了制备PROLEUKINTM产品的过程的详细描述。
发明简述
本发明的一个方面是制备在用于对需要IL-2免疫疗法的患者进行肠胃外施用的液体药物组合物中高度稳定的阿地白介素的方法,所述方法一般包括以下步骤:a)发酵用经工程化以高表达阿地白介素基因的表达载体转染的大肠杆菌(E.coli)菌株,b)破碎细菌,c)收集含有阿地白介素聚集体的包涵体,d)使用SDS清洁剂溶解阿地白介素聚集体,e)用氧化性化合物(例如氯化铜)氧化,f)陶瓷羟基磷灰石色谱,g)用有机腈(例如乙腈)稀释,h)C4柱HPLC色谱,i)透析过滤(diafiltration),以及j)过滤灭菌。
本发明的第二方面是在药学上适合的水性载剂中的阿地白介素的组合物,用于给需要IL-2免疫疗法的患者肠胃外施用,其中所述阿地白介素已使用本发明的制备方法制备,其确保阿地白介素在药物液体组合物中稳定达至少一年。用本发明的阿地白介素的组合物可制备在一个或多个预填充注射器中始终稳定的药物产品,每个注射器含有适合给定治疗的剂量,包装在盒中。该产品在注射至患者前不需要任何操作,避免了例如微生物或其它可能的污染。另一方面,可有利地引入本发明的稳定的液体阿地白介素制剂并将其均匀分布在通常在医学中用于输注、输液或临床营养的肠胃外溶液中。最近,开发出了由给定药物加阿地白介素组成的治疗(例如CEPLENETM(组胺二盐酸盐)与低剂量的proleukin联合施用以维持急性髓性白血病患者的首次缓解)。对于此类治疗,本发明的稳定的液体阿地白介素制剂可用作精制独特的组合制剂或组合试剂盒的基础。Proleukin也已用于诱导待用于细胞治疗程序的易感细胞的体外增殖(5)。对于这些程序,为了避免可以导致微生物污染的操作并且也为了获得阿地白介素在细胞培养物中的快速且均匀的分布,以低浓度配制的本发明的稳定的液体阿地白介素可以是非常有利的。这些仅为很少的实例,然而,本领域技术人员可以容易地想到本发明的稳定的液体阿地白介素制剂的其它应用。
本文的IL-2分子包括本公开的IL-2变体,其包含与IL-2氨基酸序列(SEQ ID NO:1)具有至少60%、至少61%、至少62%、至少63%、至少64%、至少65%、至少66%、至少67%、至少68%、至少69%、至少70%、至少71%、至少72%、至少73%、至少74%、至少75%、至少76%、至少77%、至少78%、至少79%、至少80%、至少81%、至少82%、至少83%、至少84%、至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%的序列相同性的氨基酸序列。这些变体包括包含具有N88R突变的氨基酸序列的IL-2变体,所述变体与野生型IL-2氨基酸序列(即SEQ ID NO:1)具有至少60%、至少61%、至少62%、至少63%、至少64%、至少65%、至少66%、至少67%、至少68%、至少69%、至少70%、至少71%、至少72%、至少73%、至少74%、至少75%、至少76%、至少77%、至少78%、至少79%、至少80%、至少81%、至少82%、至少83%、至少84%、至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%的序列相同性。实施方式还包括优先刺激Treg细胞并且包含具有N88R和C125S突变的氨基酸序列的IL-2变体,所述氨基酸序列与野生型IL-2氨基酸序列(SEQ IDNO:1)具有至少60%、至少61%、至少62%、至少63%、至少64%、至少65%、至少66%、至少67%、至少68%、至少69%、至少70%、至少71%、至少72%、至少73%、至少74%、至少75%、至少76%、至少77%、至少78%、至少79%、至少80%、至少81%、至少82%、至少83%、至少84%、至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%或至少98%的序列相同性。实施方式还包括优先刺激Treg细胞并且包含与野生型IL-2氨基酸序列(SEQ ID NO:1)具有至少60%、至少61%、至少62%、至少63%、至少64%、至少65%、至少66%、至少67%、至少68%、至少69%、至少70%、至少71%、至少72%、至少73%、至少74%、至少75%、至少76%、至少77%、至少78%、至少79%、至少80%、至少81%、至少82%、至少83%、至少84%、至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%的序列相同性的IL-2变体。
附图简述
图1:重组人白介素2(阿地白介素)的氨基酸序列(SEQ ID NO:1)。
图2:通过动态光散射测量的PROLEUKINTM和通过本发明的方法制备的阿地白介素之间的阿地白介素/SDS颗粒大小范围的比较。
图3:在0-8℃存储0个月、6个月或12个月后,通过本发明的方法制备的阿地白介素的阿地白介素/SDS颗粒大小范围。
发明详述
本发明的实施方式涉及液体配制物中高度稳定的IL-2和产生所述稳定的IL-2的方法。
定义
本文所使用的术语学仅出于描述特定实施方式的目的,并且不旨在限制本发明。除非另外特别定义,本文使用的所有技术和科学术语应被认为具有与本领域(例如细胞培养、分子遗传学和生物化学)普通技术人员通常理解的含义相同的含义。
除非上下文另外明确指出,如本文所使用的单数形式“一(a,an)”和“所述(the)”也旨在包括复数形式。此外,就在详细描述和/或权利要求中使用术语“包括(including,includes)”和“具有(having,has,with)”或它们的变型而言,此类术语旨在以类似于术语“包含”的方式表示是包含性的。
术语“约”或“大约”表示在如本领域普通技术人员确定的特定值的可接受误差范围内,其部分取决于如何测量或确定所述值,即测量系统的限制。例如,根据本领域的实践,“约”可表示在1个或多于1个标准差内。或者,“约”可表示给定值或范围的至多20%、至多10%、至多5%或至多1%的范围。或者,特别是关于生物系统或过程,该术语可表示在值的数量级以内,5倍以内,也可以是2倍以内。本申请和权利要求书中描述特定值时,除非另有说明,应假设术语“约”表示在所述特定值的可接受的误差范围内。
本文所使用的“生物培养基”是用于体外生长、培养和维持器官、组织、细胞等的任何类型的培养基。生物培养基还涵盖任何生物相容性物质(agent),任何药物赋形剂,药学上和生理上可接受的流体,如作为载剂的水、生理盐水、平衡盐溶液、水性葡萄糖、甘油等,组织或器官培养基,可给受试者体内施用的任何物质,可在测定中使用或用于稀释或维持生物学样品(例如核酸、肽等)的任何物质。
本文所使用的术语“细胞”包括原核和真核细胞。在一个实施方式中,本发明的细胞是细菌细胞。在另一实施方式中,本发明的细胞是真菌细胞,如酵母细胞。在另一实施方式中,本发明的细胞是脊椎动物细胞,例如鸟类细胞或哺乳动物细胞。在优选的实施方式中,本发明的细胞是鼠细胞或人细胞。本文所使用的术语“工程化的”(如在工程化的细胞中)是指其中导入了例如编码IL-2蛋白(例如IL-2的剪接和/或非剪接形式)的核酸分子或其片段的细胞。
本文所使用的术语“无细胞组合物”是指不含完整细胞的分离的组合物。无细胞组合物的实例包括细胞提取物和含有分离的蛋白质的组合物。
本文所使用的涉及所定义或描述的项目、组合物、装置、方法、过程、系统等的要素的术语“包含(comprising,comprise或comprised)”及其变型表示是包含性的或开放式的,允许额外的要素,从而表明所定义或描述的项目、组合物、装置、方法、过程、系统等包括那些指定的要素(或酌情包括其等同物)并且其它要素可被包括在内,但仍在所定义的项目、组合物、装置、方法、过程、系统等的范围/定义内。
术语“表达载体”是指含有编码能够被转录的基因产物的至少部分的核酸序列的载体。在一些情况下,当转录产物是mRNA分子时,其继而被翻译为蛋白质、多肽或肽。
本文所使用的术语人重组IL-2(rhIL-2)是由微生物产生的未糖基化的蛋白质,所述微生物用编码人IL-2的DNA序列或其非大幅修饰体转染。实际上,在临床实践中最常使用的IL-2人重组版本是具有以下修饰的天然人IL-2氨基酸序列的修饰版本(称为阿地白介素(PROLEUKINTM)):a)阿地白介素不具有天然IL-2中存在的N末端丙氨酸,b)阿地白介素在氨基酸125位由丝氨酸取代半胱氨酸。图1中示出了阿地白介素氨基酸序列。美国专利4518584A和相关的非美国专利中描述了阿地白介素基因的构建。美国FDA已批准阿地白介素(PROLEUKINTM)用于治疗患有转移性肾细胞癌和转移性黑素瘤的成人。PROLEUKINTM以20个用来静脉施用的一次性瓶中的无菌的白色至灰白色冻干饼形式提供。当用1.2mL注射用无菌水(USP)复水时,每mL含有1800万国际单位(1.1mg)PROLEUKINTM、50mg甘露醇和0.18mg十二烷基硫酸钠,用约0.17mg磷酸二氢钠和0.89mg磷酸氢二钠缓冲至pH 7.5(范围为7.2-7.8)。如“PROLEUKINTM(阿地白介素)注射标签-FDA”(参考ID:3165255)的重构和稀释说明”所述,复水的或稀释的PROLEUKINTM在冷藏和室温2-25℃(36-77°F)下可稳定长达48小时。
本文所使用的术语“白介素2”(IL-2)表示调节白细胞(主要是淋巴细胞)的活性的细胞因子。该细胞因子是对微生物感染的免疫应答的一部分,并通过结合至淋巴细胞表面上存在的特定受体而发挥其作用。关于IL-2的更多信息可在其“GeneCard”(登录号GC04M123831)中找到。其它登录号包括:HGNC:6001Entrez Gene:3558Ensembl:ENSG00000109471OMIM:147680UniProtKB:P60568。IL-2蛋白包括天然IL-2蛋白以及变体IL-2蛋白。无论从天然来源纯化或使用重组技术制备,本文所使用的“天然”或“野生型”IL-2序列是指人IL-2序列(例如登录号NP 000577.2)。在一些实施方式中,野生型IL-2序列包括PROLEUKINTM(阿地白介素)序列:(SEQ ID NO:1)
本文所使用的术语“试剂盒”是指用于递送材料的任何递送系统。术语“试剂盒”包括用于研究和临床应用的试剂盒。在反应测定的背景下,此类递送系统包括使反应试剂(例如适当的容器中的细胞因子、寡核苷酸、酶等)和/或支持材料(例如缓冲液、用于进行测定的书面说明书等)能够存储、从一个位置运输或递送至另一位置的系统。例如,试剂盒包括一个或多个含有相关反应试剂和/或支持材料的外壳(enclosure)(例如盒)。本文所使用的术语“分散的(fragmented)试剂盒”是指包括两个或多个单独容器的递送系统,每个容器含有全部试剂盒成分的一部分。所述容器可一起或分开地递送至预期的接收者。例如,第一容器可含有在测定中使用的酶,而第二容器含有寡核苷酸或脂质体。术语“分散的试剂盒”旨在涵盖含有根据联邦食品、药品和化妆品法520(e)章节规定的分析物特异性试剂(ASR)的试剂盒,但不限于此。实际上,术语“分散的试剂盒”中包括任何包含两个或多个单独的容器的递送系统,每个容器含有全部试剂盒成分的一部分。相反,“组合的试剂盒”是指在单个容器中(例如在容纳每个期望的成分的单个盒中)含有反应测定的所有成分的递送系统。术语“试剂盒”包括分散的试剂盒和组合的试剂盒。
本说明书和所附权利要求中所使用的术语“或”通常以包括“和/或”的意义采用,除非内容清楚地另外指出。
短语“药学上可接受的载体”是指用于施用治疗剂的载体。示例性载体包括盐水、缓冲盐水、葡萄糖、水、甘油、乙醇以及它们的组合。对于口服施用的药物,药学上可接受的载体包括但不限于药学上可接受的赋形剂,如惰性稀释剂、崩解剂、粘合剂、润滑剂、甜味剂、调味剂、着色剂和防腐剂。合适的惰性稀释剂包括碳酸钠和碳酸钙,磷酸钠和磷酸钙以及乳糖,而玉米淀粉和藻酸是合适的崩解剂。粘合剂可包括淀粉和明胶,而润滑剂(如果存在)通常是硬脂酸镁、硬脂酸或滑石粉。如果需要,可用诸如单硬脂酸甘油酯或二硬脂酸甘油酯的材料包覆片剂,以延迟在胃肠道中的吸收。
本文所使用的“稳定的”或“高度稳定的”涉及通过体外生物学活性、体外结构研究和体内治疗活性评价的本文呈现的配制物中的分子(例如IL-2)在长时间内的活性和/或完整性。IL-2的活性可通过任何标准测定来测量。可比较存储前和长期存储(例如至少一年)后的活性。与最初配制时的IL-2活性相比,高度稳定的IL-2具有相同或稍低的活性。
通过本文引用的登录号指示的Genbank和NCBI提交以引用的方式并入本文。本文引用的所有其它公开的参考文献、文件、手稿和科学文献均以引用的方式并入本文。在发生冲突的情况下,以本说明书(包括定义)为准。此外,材料、方法和实施例仅为说明性的,并且不旨在限制。
范围:在整个本公开中,可以以范围格式来表现本发明的各个方面。应理解范围格式的描述仅为方便和简洁,并且不应被解释为对本发明范围的严格限制。因此,应将范围的描述视为具体公开了所有可能的子范围以及该范围内的各数值。例如,对范围如1-6的描述应被视为具体公开了1-3、1-4、1-5、2-4、2-6、3-6等的子范围,以及该范围内的单个数例如1、2、2.7、3、4、5、5.3和6。这与范围的宽度无关。
产生稳定的IL-2配制物的方法
本发明部分地基于用于制备阿地白介素的新方法,其产生满足PROLEUKINTM的所有结构和生物学特征的产物,但令人惊讶地,如通过“体外”生物学活性、“体外”结构研究和“体内”治疗活性所评价的,其在溶液中非常稳定(至少一年)。制备在溶液中大为稳定的阿地白介素的示例性方法包括以下步骤:
a)发酵用经工程化以高表达阿地白介素基因的表达载体转染的大肠杆菌菌株。细菌是用于产生IL-2的优选微生物,并且在细菌中最优选大肠杆菌菌株。大肠杆菌B在生命科学实验室和生物技术工业中用作研究模型并且也用于蛋白质表达。诸如蛋白酶缺乏、高水平葡萄糖下低的醋酸盐产量以及增强的通透性(可能由于简单的细胞表面)等特征使大肠杆菌B成为用于生产基因工程化蛋白的期望的宿主。B菌株和K12之间的差异包括BL21(DE3)中不存在鞭毛成分基因、DNA胞嘧啶甲基化酶dcm和ompT。B菌株可能具有K12中未发现的额外的II型分泌系统。BL21(DE3)还携带DE3重组噬菌体,其带有T7 RNA聚合酶基因可指导T7启动子控制下的所克隆的基因高水平表达。用于重组蛋白表达的典型大肠杆菌菌株为:BL21(保护靶蛋白免受lon和ompT蛋白酶影响的大肠杆菌B菌株)及其衍生物例如溶源性DE3(基于T7聚合酶)、pLysS、pLysE(表达降低目标基因的基础表达的T7溶菌酶)、Origami(能够在大肠杆菌细胞质中形成二硫键)、Rosetta(增强含有大肠杆菌中很少使用的密码子的蛋白质的表达)。在K12大肠杆菌遗传背景下存在类似的形式。用于在大肠杆菌中高表达重组蛋白的典型质粒载体为:基于pBR322起点和T7/lac启动子的pET系列;具有araBAD启动子和pUC起点的pBad;具有tac启动子和pBR322起点的pGEX。融合标签序列、蛋白酶切割位点、选择标志物和菌株相容性的组合是高表达质粒变体最常见的列表的来源。
表达载体是商业可得的,并且编码阿地白介素氨基酸序列的DNA序列插入载体中。本测定中使用的细胞可为真核来源或原核来源的。例如,在一个实施方式中,所述细胞是细菌细胞。在另一实施方式中,所述细胞是真菌细胞,例如酵母细胞。在另一实施方式中,所述细胞是脊椎动物细胞,例如鸟类细胞或哺乳动物细胞。在另一实施方式中,所述细胞是人细胞。本发明的细胞可表达内源性IL-2或其片段,或可被工程化以表达内源性IL-2或其片段。例如,可通过将编码所述蛋白质的表达载体导入细胞来生产经工程化以表达IL-2或其片段的细胞。
在某些实施方式中,所述细胞是大肠杆菌细胞。可转染不同的大肠杆菌菌株以获得最佳的阿地白介素生产。
a)可在合适的生长培养基中培养产生阿地白介素的细菌。例如,培养基可包含每9升,216克酵母提取物、108克大豆蛋白胨、113克K2HPO4、20.8克KH2PO4、36ml甘油和4ml消泡剂(2%v/v)。发酵条件可为:温度:37℃±0.5℃;搅拌:350rpm±10rpm;空气流:9L/min±1L/min;pO2:设定点40%;搅拌级联:350rpm-440rpm运行21%-36%;pH 6.95-7.5;氧气流开始前入口气压2bar。此后,应遵循补料(feeding)程序。例如,通过滴加用40%p/v的葡萄糖溶液补料,以保持0.1%的浓度。OD600达到5-10后,应添加适当诱导剂如异丙基-β-D-1-硫代半乳糖苷(IPTG)以达到操作浓度。此时,可减少用葡萄糖补料,以保持约0.01%的葡萄糖浓度。通常可以在接种后约18小时-24小时停止发酵。发酵后,可通过离心或切向过滤将细菌浓缩5-7倍,并立即进行处理或在2-8℃保存(不超过24小时)或在-20℃保存(超过24小时)。
b)培养后,阿地白介素位于细菌内部,主要呈聚集体形式,称为包涵体(Ib)。可通过破碎细菌(例如通过超声处理)来分离这些Ib。为此,可在开始破碎过程之前在17-22℃之间的经纯化的水中悬浮细菌。此后,悬浮液可在破碎仪约1400bar的压力下循环2-3次。裂解液应立即处理或在-20℃保存。通过离心或切向过滤将Ib与裂解物的其它成分分离并清洗。用于Ib清洗的最终缓冲液可为TE(10mM Tris-HCl,1mM EDTA二钠,pH8.0)。Ib制剂应存储在-20℃直至进一步处理。然后将Ib悬浮在含有10mM磷酸钠、1%SDS、pH8.0的缓冲液中,并搅拌1小时。然后,加入氯化铜的氧化溶液以达到100μM的终浓度,并且应继续搅拌2小时。此后应添加EDTA以达到100μM的终浓度。混合物应在2-8℃存储,直至用于下游过程。
c)第一色谱使用陶瓷羟基磷灰石(I型80μm Bio-Rad)柱进行。加载阿地白介素制剂,用含100mM磷酸钠、0.3%SDS的pH 6.5的溶液清洗,并用含250mM磷酸钠、0.3%SDS的pH6.5的溶液洗脱。
d)第二色谱使用HPLC C4柱进行。将从第一色谱中回收的含有阿地白介素的溶液与乙腈混合(9份色谱I合并洗脱液和1份乙腈)。加载后使用乙腈梯度洗脱阿地白介素。在含有73mM磷酸钠、0.3%SDS的pH 7.4的溶液中收集含有阿地白介素的级分。
e)阿地白介素纯化的最后一步是使用5kD盒的透析过滤。使用含有0.44mg/mlSDS、50mg/ml甘露醇、1.19mg/ml磷酸氢二钠、0.26mg/ml二水合磷酸二氢钠的pH 7.5的缓冲液对阿地白介素进行平衡。
令人惊讶地发现,通过本发明的制备方法获得的阿地白介素/SDS复合物不需要如EP 1 688 146B1中所述冻干以使其稳定,因为如实施例4和实施例6中所述,它们在溶液中稳定达至少一年。
根据实施例3和实施例4中所示的动态光散射测定,通过本发明的制备方法获得的阿地白介素/SDS聚集体具有4nm-18nm之间的宽的大小范围,在约8nm处具有峰。即使复水的PROLEUKINTM也显示出相似的分布曲线,该曲线下的面积明显低于通过本发明的方法制备的阿地白介素/SDS颗粒的曲线下的面积。由于两种情况下使用的蛋白质量相同,该结果提示,复水后部分PROLEUKINTM可能保持为大的聚集体。实际上,为测量PROLEUKINTM的完全活性,据报道冻干的制剂应悬浮在SDS溶液中以避免聚集体形成(6)。
WO 2017068031A1描述了可以获得适合注射至患者的液体药物组合物,其基本上由浓度为10万-2000万IU/mL的白介素-2组成。为保持白介素2在溶液中的稳定性,该组合物应含有阴离子表面活性剂,例如十二烷基硫酸钠(SDS),其可以以约0.05mg/ml-0.5mg/ml的浓度存在。然而,如在PROLEUKINTM产品中所见,在溶液中添加SDS与IL-2形成具有生物学活性的微聚集体,并且IL-2的微聚集状态对于治疗功效是重要的(EP 1 688 146B1)。因此,应评估任何新型IL-2组合物的微聚集状态和治疗价值。关于此,实施例3和实施例4示出了微聚集状态和稳定性,并且实施例6是评估通过本发明的方法获得的阿地白介素/SDS聚集体的治疗价值的测定。
下列实施例进一步说明了本发明。这些实施例不旨在以任何方式限制本发明。
实施例
实施例1:通过本发明的生产方法获得的阿地白介素的氨基酸序列。
通过本方法中使用的表达质粒中存在的阿地白介素基因的DNA测序,推导出通过本发明的生产方法获得的阿地白介素的氨基酸序列。图1示出了所获得的氨基酸序列。
实施例2:通过本发明的生产方法获得的阿地白介素/SDS聚集体的比活性。
通过测定HT-2细胞系的增殖来确定通过本发明的生产方法获得的阿地白介素/SDS聚集体的生物学活性。用白介素2(人,rDNA衍生的)的WHO国际标准(NIBSC代码:86/500)校准的20个独立批次(lot)的比活性在16.6IU/mg-19.5IU/mg范围内。该比活性范围与根据EP 1 688 146B1制备的阿地白介素/SDS聚集体的宣称的比活性相似。
实施例3:通过动态光散射测量,从冻干的商业制剂复水后获得的SDS/Proleukin聚集体与使用本发明的方法获得的SDS/阿地白介素聚集体的大小相当。
使用Zetasizer Nano ZS和Zeta Nano系列软件(Malvern Instruments,德国),使用动态光散射测定阿地白介素/SDS聚集体的大小和多分散性。将100μL样品稀释在1mL纯水中,并在25℃测量5次,每次持续30秒,测量之间的平衡时间为30秒。颗粒大小表示为以nm计的流体动力学直径。图2示出了用本发明的方法获得的SDS/阿地白介素聚集体和从冻干的商业制剂复水后的PROLEUKINTM的动态光散射模式。通过本发明的生产方法获得的阿地白介素/SDS聚集体显示出4nm-18nm之间的宽的大小范围,在约8nm处具有峰。PROLEUKINTM也显示了类似的分布曲线。然而,该曲线下的面积明显低于通过本发明的方法制备的阿地白介素/SDS颗粒的曲线下的面积。由于分析的两种样品中使用的蛋白质量相同,该结果提示,复水后部分PROLEUKINTM可能保持为大的聚集体,不适合用所使用的技术进行研究。
实施例4:通过本发明的生产方法获得的阿地白介素的SDS/阿地白介素制剂的稳定性
表1示出了比活性随在2-8℃存储时间的活性变化。除比活性外,测定了各样品中的以下特征:对异常现象的肉眼观察、pH、体积、蛋白质印迹、SDS PAGE(还原和非还原条件)、Lowry、内毒素污染(LAL测试)和无菌性。在所有测量的时间,这些测定结果正常。
表1:通过本发明的方法获得的可溶性阿地白介素/SDS聚集体的比活性
此外,图3显示在2-8℃存储6个月或12个月后,通过动态光散射测量的阿地白介素/SDS聚集体的大小分布在本发明的阿地白介素液体组合物中被很好地保留。
实施例5:与通过本发明的生产方法获得的SDS/阿地白介素聚集体一起温育的人外周血单个核血细胞(PMBC)中的调节性T细胞(Treg)扩增
使用DYNABEADSTM调节性CD4+/CD25+T细胞试剂盒(ThermoFisher Scientific)从人PMBC回收Treg细胞。使用CD3/CD28人Treg扩增剂(ThermoFisherScientific)方案评价阿地白介素/SDS聚集体诱导Treg增殖的能力。使用500IU的阿地白介素制剂进行Treg扩增。扩增7天后对Treg进行计数。使用本发明的方法或EP1 688 146B1中完全公开的方法获得的阿地白介素/SDS聚集体的扩增均为76-125倍。
实施例6:使用本发明的生产方法获得的阿地白介素/SDS聚集体的治疗活性
为比较通过本发明的方法制备的阿地白介素聚集体和PROLEUKINTM的阿地白介素/SDS聚集体的治疗效率,使用了如(7)所述使用B16F10肿瘤细胞的肺转移模型。向8周龄的雌性C57BL/6小鼠尾静脉静脉内注射0.5ml含有2×105个B16细胞的细胞悬液。此后,将小鼠分为3组,每组10只。第1组小鼠从第3天至第10天每天接受7.0mg/Kg剂量的PROLEUKINTM。第2组小鼠从第3天至第10天每天接受7.0mg/Kg剂量的通过本发明方法制备的阿地白介素微聚集体。第3组小鼠从第3天至第10天每天接受载剂。在肿瘤接种后第16天对小鼠实施安乐死,并如(7)所述对肺转移进行计数。表2显示通过本发明的方法制备的微聚集体观察到的转移的中位数为4.3(范围1-12),相比之下PROLEUKINTM为5.5(范围3-22),该差异不显著。
表2:使用本发明的生产方法获得的阿地白介素/SDS聚集体的治疗活性
通过本发明的方法制备并用于该测定的阿地白介素是新生产的。与此不同的是,表3示出了在2-8℃存储6个月或12个月后对通过本发明的方法制备的阿地白介素进行的相同测定。
表3:在2-8℃储存6个月或12个月后,使用本发明的生产方法获得的阿地白介素/SDS聚集体的治疗活性。这些结果加强了通过本发明的方法获得的阿地白介素的液体制剂稳定达至少一年的观点。
序列
SEQ ID NO:1
长度:132
PRT
生物体:人工序列,人白细胞介素2的成熟的des-alanyl-1丝氨酸125变体
参考文献
美国专利文件
US 4,604,377 1986年8月5日
其它专利文件
EP 1688146 B1 2007年7月18日
WO 2 017 068 031 A1 2017年4月27日
其它出版物
1-Malek TR.The biology of interleukin-2.Annu Rev Immunol.(2008).26:453-579.
DOI:10.1146/annurev.immunol.26.021607.090357
2-Dudley ME et al.Adoptive Cell Transfer Therapy FollowingNonMyeloablative but Lymphodepleting Chemotherapy for the Treatment ofPatients With Refractory Metastatic Melanoma.Journal of Clinical Oncology.(2005).23:2346-57.DOI:
10.1200/JCO.2005.00.240
3-Langerman A,Callender GG,Nishimura MI.Retroviral transduction ofpeptide stimulated t cells can generate dual t cell receptor-expressing(bifunctional)t cells reactive with two defined antigens.Journal ofTranslational Medicine.(2004).2:4-8.DOI:10.1186/1479-5876-2-42
4-Magee MS,Snook AE.Challenges to chimeric antigen receptor(CAR)-Tcell therapy for cancer.Discov Med.(2014).18:265-71
5-Arenas-Ramirez N,Woytschak J,Boyman O.Interleukin-2:Biology,Designand Application.Trends Immunol.(2015).36:763-777.DOI:10.1016/j.it.2015.10.003
6-Hank JA,Surfus J,Gan,Albertini M,Lindstrom M,Schiller JH,Hotton KM,Khorsand M,Sondel PM.Distinct clinical and laboratory activity of tworecombinant interleukin-2preparations.Clin Cancer Res.19995:281-9
7-Overwijk WW,Restifo NP.B16as a Mouse Model for Human Melanoma.CurrProtoc Immunol.(2001).CHAPTER:Unit-20.1.
Claims (12)
1.一种生产阿地白介素的方法,所述阿地白介素适合于制备包含药物水性载剂中的阿地白介素的组合物,所述方法包括:
将用编码阿地白介素基因的表达载体转染的细菌细胞进行发酵;
破碎所述细菌细胞;
收集含有阿地白介素的包涵体;
通过清洁剂使所述包涵体溶解,然后进行氧化和第一色谱;
用有机腈稀释并且进行第二色谱;
对所述阿地白介素进行透析过滤和灭菌。
2.权利要求1的方法,其中所述细菌细胞是大肠杆菌(Escherichia coli)细菌细胞。
3.权利要求1的方法,其中所述清洁剂是十二烷基硫酸钠(SDS)。
4.权利要求1的方法,其中所述氧化是用氧化性化合物进行的。
5.权利要求1的方法,其中所述第一色谱是陶瓷羟基磷灰石色谱。
6.权利要求1的方法,其中所述第二色谱是高效液相色谱(HPLC)。
7.权利要求6的方法,其中所述HPLC是C4柱HPLC色谱。
8.权利要求1的方法,其中通过体外生物学活性测定测量,所述阿地白介素在所述药物液体组合物中保持稳定达至少一年。
9.权利要求1的方法,其中通过动态光散射测量,所述阿地白介素在所述液体组合物中包含稳定的阿地白介素/SDS聚集体分布达至少一年,所述分布在约4nm-18nm范围内,具有约8nm处的峰。
10.权利要求1的方法,其中在人癌症的动物模型中测量,所述阿地白介素在所述液体组合物中具有稳定的治疗活性达至少一年。
11.权利要求1的方法,其中所述阿地白介素诱导细胞培养物中的人调节性T细胞增殖。
12.一种含有通过权利要求1的方法获得的阿地白介素的药物组合物,其中所述阿地白介素:a)通过体外生物学活性测定测量,在所述药物液体组合物中保持稳定至少一年,b)通过动态光散射测量,在所述液体组合物中具有稳定的阿地白介素/SDS聚集体分布至少一年,所述分布在约4nm-18nm范围内,具有约8nm处的峰,c)在人癌症的动物模型中测量,在所述液体组合物中具有稳定的治疗活性达至少一年,以及d)足以扩增细胞培养物中的人调节性T细胞。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201862684288P | 2018-06-13 | 2018-06-13 | |
US62/684,288 | 2018-06-13 | ||
PCT/US2019/037012 WO2019241534A1 (en) | 2018-06-13 | 2019-06-13 | Method to prepare therapeutically active aldesleukin highly stable in liquid pharmaceutical compositions |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113166221A true CN113166221A (zh) | 2021-07-23 |
Family
ID=68841872
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201980039901.2A Pending CN113166221A (zh) | 2018-06-13 | 2019-06-13 | 制备在液体药物组合物中高度稳定的具有治疗活性的阿地白介素的方法 |
CN202110049022.7A Pending CN114601917A (zh) | 2018-06-13 | 2021-01-14 | 液体药物组合物中高度稳定的具有治疗活性的阿地白介素 |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110049022.7A Pending CN114601917A (zh) | 2018-06-13 | 2021-01-14 | 液体药物组合物中高度稳定的具有治疗活性的阿地白介素 |
Country Status (9)
Country | Link |
---|---|
US (4) | US20210299222A1 (zh) |
EP (2) | EP3807305A4 (zh) |
JP (2) | JP2021528101A (zh) |
KR (2) | KR20210021357A (zh) |
CN (2) | CN113166221A (zh) |
CA (2) | CA3103603A1 (zh) |
IL (2) | IL279259A (zh) |
SG (1) | SG11202012299SA (zh) |
WO (1) | WO2019241534A1 (zh) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
SG11202012299SA (en) * | 2018-06-13 | 2021-01-28 | Akron Biotechnology Llc | Method to prepare therapeutically active aldesleukin highly stable in liquid pharmaceutical compositions |
KR20230169147A (ko) * | 2021-03-11 | 2023-12-15 | 더 메서디스트 하스피틀 | 질환 치료를 위한 방법 및 조성물 |
WO2023072858A1 (en) * | 2021-10-25 | 2023-05-04 | Ellennbe Gmbh | Pharmaceutical composition and kit comprising an immunomodulatory substance for treating diseases |
WO2024173571A1 (en) * | 2023-02-14 | 2024-08-22 | Amcyte Pharma, Inc. | Interleukin formulations for in vivo use |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1688146A1 (en) * | 2005-02-07 | 2006-08-09 | Chiron Corporation | Preparing aldesleukin for pharmaceutical use |
WO2017068031A1 (en) * | 2015-10-22 | 2017-04-27 | Iltoo Pharma | Pharmaceutical compositions of il-2 |
Family Cites Families (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4452773A (en) | 1982-04-05 | 1984-06-05 | Canadian Patents And Development Limited | Magnetic iron-dextran microspheres |
US4518584A (en) | 1983-04-15 | 1985-05-21 | Cetus Corporation | Human recombinant interleukin-2 muteins |
US4569790A (en) * | 1984-03-28 | 1986-02-11 | Cetus Corporation | Process for recovering microbially produced interleukin-2 and purified recombinant interleukin-2 compositions |
US4604377A (en) | 1984-03-28 | 1986-08-05 | Cetus Corporation | Pharmaceutical compositions of microbially produced interleukin-2 |
US4795698A (en) | 1985-10-04 | 1989-01-03 | Immunicon Corporation | Magnetic-polymer particles |
WO1990007380A2 (en) | 1988-12-28 | 1990-07-12 | Stefan Miltenyi | Methods and materials for high gradient magnetic separation of biological materials |
US5200084A (en) | 1990-09-26 | 1993-04-06 | Immunicon Corporation | Apparatus and methods for magnetic separation |
US5827642A (en) | 1994-08-31 | 1998-10-27 | Fred Hutchinson Cancer Research Center | Rapid expansion method ("REM") for in vitro propagation of T lymphocytes |
US6955807B1 (en) * | 1998-05-15 | 2005-10-18 | Bayer Pharmaceuticals Corporation | IL-2 selective agonists and antagonists |
WO2001024814A1 (en) * | 1999-10-04 | 2001-04-12 | Chiron Corporation | Stabilized liquid polypeptide-containing pharmaceutical compositions |
GB2415904B (en) * | 2005-02-07 | 2006-10-25 | Chiron Corp | Preparing aldesleukin for pharmaceutical use |
ES2318916B1 (es) * | 2005-02-07 | 2010-03-22 | Chiron Corporation | Procedimiento para preparar aldesleukina para uso farmaceutico. |
EP3338895B1 (en) | 2007-12-07 | 2022-08-10 | Miltenyi Biotec B.V. & Co. KG | Sample processing systems and methods |
US20120164718A1 (en) | 2008-05-06 | 2012-06-28 | Innovative Micro Technology | Removable/disposable apparatus for MEMS particle sorting device |
GB201515244D0 (en) * | 2015-08-27 | 2015-10-14 | Univ Birmingham | Treatment of inflammatory disease or condition |
SG11202012299SA (en) * | 2018-06-13 | 2021-01-28 | Akron Biotechnology Llc | Method to prepare therapeutically active aldesleukin highly stable in liquid pharmaceutical compositions |
-
2019
- 2019-06-13 SG SG11202012299SA patent/SG11202012299SA/en unknown
- 2019-06-13 WO PCT/US2019/037012 patent/WO2019241534A1/en unknown
- 2019-06-13 JP JP2021519521A patent/JP2021528101A/ja active Pending
- 2019-06-13 EP EP19819023.3A patent/EP3807305A4/en active Pending
- 2019-06-13 KR KR1020217000804A patent/KR20210021357A/ko not_active Application Discontinuation
- 2019-06-13 CA CA3103603A patent/CA3103603A1/en active Pending
- 2019-06-13 CN CN201980039901.2A patent/CN113166221A/zh active Pending
-
2020
- 2020-12-07 IL IL279259A patent/IL279259A/en unknown
- 2020-12-09 US US17/116,722 patent/US20210299222A1/en active Pending
- 2020-12-11 US US17/118,915 patent/US20210246184A1/en active Pending
- 2020-12-11 CA CA3102525A patent/CA3102525A1/en active Pending
- 2020-12-11 JP JP2020206270A patent/JP2022091653A/ja active Pending
- 2020-12-11 IL IL279389A patent/IL279389A/en unknown
-
2021
- 2021-01-14 CN CN202110049022.7A patent/CN114601917A/zh active Pending
- 2021-12-02 KR KR1020210170659A patent/KR20220081909A/ko unknown
- 2021-12-08 EP EP21213209.6A patent/EP4011388A1/en active Pending
-
2023
- 2023-06-29 US US18/344,130 patent/US20230331802A1/en active Pending
- 2023-11-27 US US18/519,193 patent/US20240141008A1/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1688146A1 (en) * | 2005-02-07 | 2006-08-09 | Chiron Corporation | Preparing aldesleukin for pharmaceutical use |
WO2017068031A1 (en) * | 2015-10-22 | 2017-04-27 | Iltoo Pharma | Pharmaceutical compositions of il-2 |
Also Published As
Publication number | Publication date |
---|---|
IL279259A (en) | 2021-01-31 |
CA3103603A1 (en) | 2019-12-19 |
US20210246184A1 (en) | 2021-08-12 |
EP3807305A1 (en) | 2021-04-21 |
KR20210021357A (ko) | 2021-02-25 |
US20230331802A1 (en) | 2023-10-19 |
EP4011388A1 (en) | 2022-06-15 |
IL279389A (en) | 2022-07-01 |
CA3102525A1 (en) | 2022-06-09 |
CN114601917A (zh) | 2022-06-10 |
SG11202012299SA (en) | 2021-01-28 |
WO2019241534A1 (en) | 2019-12-19 |
US20240141008A1 (en) | 2024-05-02 |
JP2022091653A (ja) | 2022-06-21 |
EP3807305A4 (en) | 2022-04-20 |
JP2021528101A (ja) | 2021-10-21 |
US20210299222A1 (en) | 2021-09-30 |
KR20220081909A (ko) | 2022-06-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN113166221A (zh) | 制备在液体药物组合物中高度稳定的具有治疗活性的阿地白介素的方法 | |
Zhang et al. | DP7-C-modified liposomes enhance immune responses and the antitumor effect of a neoantigen-based mRNA vaccine | |
TWI488864B (zh) | 用於治療癌症及慢性感染之具有促效劑活性之介白素-2(il-2)衍生物多肽 | |
CN116948006A (zh) | 白介素-2多肽偶联物及其用途 | |
US20050065107A1 (en) | Plasmids suitable for IL-2 expression | |
AU2015301936A1 (en) | Modified IL-2 variants that selectively activate regulatory T cells for the treatment of autoimmune diseases | |
EP3010932B1 (en) | Bacterial hyaluronidase and process for its production | |
CN101238143A (zh) | 将非天然编码氨基酸并入蛋白质中 | |
JPS60115528A (ja) | ヒトインタ―ロイキン―2蛋白質を含有する抗腫瘍用または免疫機能低下疾患治療用組成物 | |
KR20070114263A (ko) | 바실러스 안트라시스 유래의 방어 항원 제조 방법 | |
CN1134111A (zh) | 使用胰岛素样生长因子结合蛋白质的方法 | |
US20210052643A1 (en) | Modified macrophages and macrophage precursors and associated methods | |
KR20220151202A (ko) | 인터류킨-2 폴리펩타이드 접합체 및 그의 사용 방법 | |
CN114788876A (zh) | 治疗糖尿病的mRNA药物制剂及其制备方法与应用 | |
TWI818166B (zh) | 用於生產膠原蛋白7組合物之系統及方法 | |
CN110305221B (zh) | 一种增强型抗肿瘤融合蛋白及制备方法及用途 | |
CN108265044B (zh) | 聚乙二醇定点修饰的精氨酸脱亚胺酶及其制备方法与应用 | |
US6652850B1 (en) | Adeno-associated viral liposomes and their use in transfecting dendritic cells to stimulate specific immunity | |
AU2021461372A1 (en) | Long-acting recombinant human interleukin-2 fusion protein, and preparation method therefor and application thereof | |
Farnia et al. | Increased production of soluble vascular endothelial growth factors receptor-1 in CHO-cell line by using new combination of chitosan-protein lipid nanoparticles | |
JPH05103666A (ja) | ヒト神経成長因子を発現させるための方法 | |
CN112105631A (zh) | Treg耗尽和检查点抑制剂的组合 | |
TWI839476B (zh) | 介白素-2多肽共軛物及其用途 | |
RU2289623C2 (ru) | ПЛАЗМИДНАЯ ДНК pBSH2EGF, КОДИРУЮЩАЯ СИНТЕЗ ЭПИДЕРМАЛЬНОГО ФАКТОРА РОСТА ЧЕЛОВЕКА, И СПОСОБ ЕГО ПОЛУЧЕНИЯ С ПОМОЩЬЮ ДАННОЙ ДНК | |
KR20110121208A (ko) | 수지상 세포를 포함하는 신세포암의 치료용 조성물 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |